The objective of the research grant is to elucidate the molecular mechanisms involved in the biosynthesis of the plasma lipoproteins, apo E ("arginine-rich" protein) and apoA-I in the rat. The messenger RNA's for apoE and apoA-I will be isolated from the rat liver and jejunum. Their translation in a heterologous system in vitro will be studied and their products characterized. The in vitro products will also be analyzed by N-terminal sequencing. The mRNA's will be purified and the double stranded (ds) cDNAs will be synthesized in vitro using reverse transcriptase. The ds cDNAs will be amplified by insertion into the plasmid pBR322. The amplified DNAs will be characterized by restriction endonucleolytic mapping and by nucleotide sequencing. The natural genes for apoE and apoA-I will be partially purified from restriction fragments of total rat DNA, using the nick-translated cloned DNAs as hybridization probes. The respective gene fragments will be amplified and purified by insertion into a lambda phage system. The cloned natural gene fragments will be characterized by restriction mapping and nucleotide sequencing. We will look for evidence of intervening sequences in the natural genes. Using the nick-translated cloned natural gene fragments and ds cDNAs as hybridization probes, we will look for evidence of pre-mRNAs for apoE and apoA-I in nuclear RNA. We will also study these potential precursor mRNAs for evidence of transcription of intervening sequences.